The human pathogen herpes simplex virus type 2 (HSV-2) is a leading cause of genital ulcer disease and a risk factor for human immunodeficiency virus (HIV) acquisition and transmission. Developing an effective HSV-2 vaccine is a public health priority for controlling the pandemic; however success has been limited largely due to our poor understanding of protective immunity required for containing HSV-2 infection and reactivation in humans. Our work has shown that the peripheral mucosal immune system, specifically HSV specific CD8+ T cells, persist in the periphery at the dermal-epidermal junction (DEJ) contiguous to the sensory nerve endings where HSV virion particles are released, and that HSV-2 specific CD8+ T cells frequently encounter viral antigens. Recent studies have shown that over 80% of HSV-2 reactivations are subclinical and of short duration, typically lasting only 6 to 12 hours, indicating that a rapid host immune response in the genital skin and mucosa can contain local HSV reactivation thus preventing symptoms. We hypothesize that persistent CD8+ T cells at the dermal-epidermal junction exert antiviral effector functions upon re-encountering viral antigen and rapidly eliminate infected cells before the virus can cause clinically symptomatic disease. We have recently developed novel technologies to define, in situ, the mechanisms by which HSV specific CD8+ T cells contain virus in the periphery, a key concept for the rational design and development of novel immunotherapeutic as well as prophylactic vaccines against herpes simplex virus. We have developed and perfected a cell-type specific laser capture microdissection (LCM) method. Using genome transcriptional profiles of LCM-CD8+ T cells, we have shown that CD8+ T cells at the dermal-epidermal junction (DEJ CD8+) have expression profiles consistent with cytolytic activity, metabolic activation and proliferative potential. We have also applied T cell receptor (TCR) high-throughput sequencing technology to LCM-CD8+ cells to determine the breadth and clonality of the CD8 TCR repertoire in locally affected skin/mucosa tissue and in blood. These studies allow us to compare T cell populations at the dermal-epidermal junction and dermal perivascular sites, and to evaluate, at the TCR sequence level, their T cell clonotypes and dominance over the course of herpes lesion evolution. The unique ability of our group to obtain sequential biopsy samples of HSV infected genital lesions and normal tissue, and to study patient cohorts with distinct clinical phenotypes, will yield opportunities to not only increase knowledge about HSV pathogenesis, but also the kinetics and mechanistic functions of resident mucosal T cells in HSV-2 containment. This proposal is designed to define immune control mechanisms and proliferation potentials for CD8+ T cells at their original anatomic sites and native physiological state in skin and mucosa, thus characterizing the molecular interactions between HSV-2 and human host in vivo.